Analytical sandwich test for determining NT-proBNP

ABSTRACT

The present invention concerns an immunological test for determining NT-proBNP comprising at least two antibodies to NT-proBNP, wherein at least one of the antibodies to NT-proBNP is a monoclonal antibody. The epitopes recognized by the antibodies can slightly overlap.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/994,851, filed Nov. 22, 2004, now U.S. Pat. No. 7,507,550. U.S. Ser.No. 10/994,851 claims the benefit of German Patent Application SerialNo. DE 103 55 731.8.

BACKGROUND OF THE INVENTION

The present invention concerns an analytical sandwich test, inparticular a test element and in particular in the form of animmunochromatographic test strip using the sandwich principle todetermine N-terminal pro-brain natriuretic peptide (NT-proBNP).

NT-proBNP is a very promising marker for the diagnosis and management ofheart failure. At present, the only NT-proBNP test that is available onthe in-vitro diagnostic market is the fully automated Elecsys® NT-proBNPtest from Roche Diagnostics which is based on a sandwich reaction withelectrochemiluminescence detection. This test is designed to be used inlarge central laboratories and in addition to liquid reagents that haveto be exactly dosed, requires a relatively complex instrument to dosethe liquids and to detect the luminescence signal in order to carry outthe test. A simple to use, rapid test for NT-proBNP which if needed canbe evaluated visually without an evaluation instrument is presently noton the market.

In patients with acute respiratory distress it is advantageous to carryout a NT-proBNP determination as rapidly as possible in order to excludeor diagnose heart failure as a cause of the dyspnoea and to initiateappropriate treatment. Since the Elecsys® NT-proBNP test can only becarried out in a central laboratory, it is difficult to rapidlydetermine NT-proBNP outside the routine times. Hence, it would beparticularly advantageous for the emergency ward if a rapid test wereavailable which could be carried out directly in the emergency wardoutside of routine times. This rapid test should, however, ensure thesame reference ranges and cut-offs as the reference method in thecentral laboratory (Elecsys® NT-proBNP) in order to enable a goodcomparability of the results independently of the type of test that isactually carried out.

The polyclonal antibodies (PAB) used in the Elecsys® NT-proBNP testrecognize a very special fraction of NT-proBNP (“native” NT-proBNP; seeInternational Patent Application PCT/EP2004/005091 dated May 12, 2004from Klemt et al.; according to this the test recognizes the epitopes ofNT-proBNP comprising the amino acids 1-21 (AA 1-21) and 39-50 (AA39-50)). However, it has turned out that these polyclonal antibodies areunsuitable for NT-proBNP rapid tests that use particulate labels such ascolloidal gold as a label since they exhibit a high undesiredvariability in the signal generation due to physico-chemicalinteractions with the components of the rapid test (such as the supportmaterials, matrices, etc.). This results in considerable fluctuations inthe quality of the test from batch to batch.

SUMMARY OF THE INVENTION

It is against the above background that the present invention providescertain unobvious advantages and advancements over the prior art. Inparticular, the inventors have recognized a need for improvements inrapid analytical tests for determining NT-proBNP, which can bereproducibly manufactured and has a good correlation to the laboratorymethod.

In various embodiments of the invention, the following combinations ofantibodies are used:

Combination I

-   -   i. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids of human NT-proBNP selected        from the group consisting of: 1-21, 13-16, 22-38, 51-76 and        64-67;    -   ii. a polyclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 39-50;

Combination II

-   -   i. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids of human NT-proBNP selected        from the group consisting of: 1-21, 22-37, and 43-76;    -   ii. a polyclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 38-42;

Combination III

-   -   i. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids of human NT-proBNP selected        from the group consisting of: 1-21, 22-43, and 51-76;    -   ii. a polyclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 44-50;

Combination IV

-   -   i. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 27-31;    -   ii. a polyclonal antibody directed against an epitope of        NT-proBNP selected from the group consisting of: 39-50, 38-42,        and 44-50;

Combination V

-   -   i. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 13-16;    -   ii. a polyclonal antibody directed against an epitope of        NT-proBNP selected from the group consisting of: 39-50, 38-42,        and 44-50;

Combination VI

-   -   i. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 27-31 of human NT-proBNP;    -   ii. a monoclonal antibody directed against an epitope of        NT-proBNP comprising amino acids 38-42 of human NT-proBNP.

In accordance with one embodiment of the present invention, animmunological test for determining NT-proBNP is provided comprising atleast two antibodies to NT-proBNP, wherein at least one of theantibodies to NT-proBNP is a monoclonal antibody. One of theseantibodies is directed at least against parts of the epitope ofNT-proBNP comprising the amino acids 38 to 50. In addition, one of theseantibodies is directed at least against parts of the epitope ofNT-proBNP comprising the amino acids 1 to 37 or 43 to 76. The epitoperecognized by the antibodies can slightly overlap.

These and other features and advantages of the present invention will bemore fully understood from the following detailed description of theinvention taken together with the accompanying claims. It is noted thatthe scope of the claims is defined by the recitations therein and not bythe specific discussion of features and advantages set forth in thepresent description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentinvention can be best understood when read in conjunction with thefollowing drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1 shows a diagram of a rapid test device according to oneembodiment of the present invention in the form of animmunochromatographic test strip;

FIG. 2 shows the correlation of the antibody combination MAB 17.3.1(13-16)/PAB (39-50) in the Elecsys® wet test format with the Elecsys®reference method PAB (1-21)/PAB (39-50);

FIG. 3 shows the correlation of the antibody combination MAB 18.4.34(27-31)/PAB (39-50) in the Elecsys® wet test format with the Elecsys®reference method PAB (1-21)/PAB (39-50);

FIG. 4 shows function curves of NT-proBNP test strips according toExample 1 with different antibody combinations; and

FIG. 5 shows the correlation of an NT-proBNP test strip with theantibody combination: Au-MAB 18.4.34 (27-31)/Bi-PAB (39-50) to theElecsys® NT-proBNP test kit.

Skilled artisans appreciate that elements in the figures are illustratedfor simplicity and clarity and have not necessarily been drawn to scale.For example, the dimensions of some of the elements in the figures maybe exaggerated relative to other elements to help improve understandingof the embodiment(s) of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The inventive solution for producing an immunological test in a sandwichformat in accordance with one embodiment of the present invention and,in particular, a rapid test which correlates well with the Elecsys®reference method uses a combination of antibodies comprising at leasttwo antibodies to NT-proBNP, where at least one antibody is a monoclonalantibody (MAB). Another antibody of the sandwich test according to anembodiment of the present invention can either also be a MAB or apolyclonal antibody (PAB). In this connection one of these antibodies(abbreviated AB) is directed at least against parts of the epitope ofNT-proBNP comprising amino acids 38 to 50 (in the following alsoabbreviated to AB (38-50) or MAB (38-50) or PAB (38-50)). At least oneadditional antibody is directed at least against parts of the epitope ofNT-proBNP comprising amino acids 1 to 37 or 43 to 76 (in the followingabbreviated to AB (1-37) or AB (43-76) or MAB (1-37) or MAB (43-76) orPAB (1-37) or PAB (43-76)). The epitopes recognized by the antibodiescan slightly overlap, typically by less than 5 amino acids, moretypically by less than 2 amino acids.

A combination of antibodies is typical comprising at least onepolyclonal antibody (PAB) and one monoclonal antibody (MAB) (so-calledPAB/MAB combination) to NT-proBNP.

The term PAB (X-Y) as used herein means a polyclonal antibody which isdirected against the epitope of NT-proBNP comprising the amino acids Xto Y. MAB (X-Y) is a corresponding monoclonal antibody. AB (X-Y)generally denotes an antibody (e.g., PAB or MAB) which is directedagainst the epitope of NT-proBNP comprising the amino acids X to Y.

MAB a.b.c. (X-Y) is a monoclonal antibody directed against the epitopeof NT-proBNP comprising the amino acids X to Y which is obtained from adeposited cell line a.b.c.

In order to guarantee a reproducible quality of the antibody-labelconjugate, the MAB is typically immobilized on a particulate label, inparticular on a gold label. Other suitable particulate labels are forexample coloured latices, other metal sol labels, polymer labels orsemiconductor nanocrystals (so-called quantum dots). The MAB-labelconjugate is typically provided on the rapid test device in such amanner that it can be detached from it by the sample liquid, for exampleby impregnating suitable support materials such as fleeces, membranes,etc. It is, however, also possible to add the MAB-label conjugate as asolution to the rapid test.

The PAB which is typically obtained by immunizing mammals, in particularsheep, goats or rabbits, is typically provided in the rapid test as abiotin derivative and can be bound to an avidin or streptavidindetection line. However, it also possible to directly immobilize the PABin the rapid test device, for example in the form of a detection line ona suitable chromatography membrane.

According to an embodiment of the present invention, it is also possiblealthough less typical, to use the labelled AB, in particular thelabelled MAB, and the second antibody, in particular the second MAB orPAB in solution or in solutions for the rapid test. A binding partnerwhich can capture the appropriately labelled AB is then located in adetection zone on the test device and thus binds the sandwich complexcomprising first antibody, analyte and second antibody to a solid phaseof the rapid test.

The MAB used according to an embodiment of the present invention doesnot necessarily have to recognize the epitope (AA 1-21) that is detectedin the reference system (Elecsys® test) in order to ensure goodcorrelation with the reference test: the antibody combinations and, inparticular, the MAB/PAB combinations MAB 17.3.1 (13-16)/PAB (39-50) andMAB 18.4.34 (27-31)/PAB (39-50) correlate well with the Elecsys®reference system which uses polyclonal antibodies to the epitopes AA1-21 and AA 39-50 of NT-proBNP (PAB (1-21) and PAB (39-50)). Otheruseful combinations include MAB 17.3.1 (13-16)/PAB (38-42) and MAB18.4.34 (27-31)/PAB (38-42).

The polyclonal antibodies such as PAB (1-21) and PAB (39-50) can beobtained, characterized and identified by methods known to a personskilled in the art especially in analogy to example 2 of WO 00/45176.

The monoclonal antibodies such as MAB (38-42) and MAB (44-50) can beobtained, characterized and identified by methods known to a personskilled in the art especially in analogy to example 3 of WO 00/45176 orexample 3 of the International Patent Application PCT/EP2004/005091dated May 12, 2004 (Klemt et al.).

The antibodies are labelled for example with gold or other labels,biotin, etc. by methods known to a person skilled in the art (cf., alsoexample 2 in WO 00/45176 and example 2 of the International PatentApplication PCT/EP2004/005091 dated May 12, 2004 by Klemt et al.).Labelling with gold is, for example, described in detail in EP-A 0 898170.

In particular, the typical monoclonal antibodies MAB 17.3.1 (13-16), MAB16.1.39 (38-42), MAB 18.29.23 (64-67) and MAB 18.4.34 (27-31) can beobtained according to example 3 of the International Patent ApplicationPCT/EP2004/005091 dated May 12, 2004 by Klemt et al. Corresponding celllines are deposited at the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSZM), Mascheroder Weg 1B, D-38124 Bruanschweig,Germany (accession numbers of the depository and date of deposition: DSMACC2591 (7^(th) May 2003) for MAB 17.3.1 (13-16); DSM ACC2590 (7^(th)May 2003) for MAB 16.1.39 (38-42); DSM ACC2593 (7^(th) May 2003) for MAB18.29.23 (64-67) and DSM ACC2592 (7^(th) May 2003) for MAB 18.4.34(27-31).

The combination of MAB 18.4.34 (27-31)/PAB (39-50) results in arelatively good correlation with the reference test as does thecombination MAB 17.3.1 (13-16)/PAB (39-50) (see also example 2).

In addition the combination MAB 18.4.34 (27-31)/PAB (39-50) proved to beparticularly advantageous for the test according to an embodiment of thepresent invention: this combination enabled a function curve to beadapted that is particularly suitable for rapid tests (see example 3).In comparison, the combination MAB 17.3.1 (13-16)/PAB (39-50) exhibiteda poorer test sensitivity.

In order that the invention may be more readily understood, reference ismade to the following examples, which are intended to illustrate theinvention, but not limit the scope thereof.

EXAMPLES Example 1 Preparation of a Test Device for DeterminingNT-proBNP from Whole Blood

The test device (FIG. 1) consists of a support material (5) on which asample application zone (1), an erythrocyte separation zone (2), adetection zone (3) and a suction zone (4) are mounted. A sampleapplication matrix (6) which partially overlaps an erythrocyteseparation zone (7) is located in the sample application zone (1). Theerythrocyte separation matrix (7) in turn slightly overlaps thedetection matrix (8) (detection zone) on which an immobilized substanceis applied in the form of a line (9). A suction matrix (10) slightlyoverlaps the detection matrix (8). All reagents that are necessary toform a complex with the analyte to be detected are accommodated in thesample application matrix (6). For example, the sample application zonecan be composed of two fleeces on top of one another where the first(“gold fleece”) is impregnated with a gold-labelled antibody toNT-proBNP (MAB 18.4.34 (27-31)) and the second fleece (“biotin fleece”)contains a biotinylated antibody to NT-proBNP (PAB (39-50)). A line (9)made of streptavidin is applied within the detection zone.

A polyester foil (Pütz) of 350 μm thickness is used as the support layer(5). A polyester fleece (Roche Diagnostics) of 360 μm thickness is usedas the “gold fleece” or “biotin fleece” of the sample application matrix(6). A glass fibre fleece (Roche Diagnostics) of 1.8 mm thickness isused as an erythrocyte separation matrix (7). A nitrocellulose membrane(Sartorius) of 140 μm thickness is used as the detection matrix (8). Aglass fibre fleece (Roche Diagnostics) of 1.8 mm thickness is used asthe suction matrix (10). The individual components (6, 7, 8, 10) areglued slightly overlapping on the support layer (5) by means of hot-meltadhesive as shown in FIG. 1.

The impregnation formulation of the “gold and biotin fleeces” is:

“biotin fleece”: 100 mM Hepes pH 7.4, 0.1% Tween ®, 20 μg/mlbiotinylated PAB (39-50) “gold fleece”: 100 mM Hepes pH 7.4, OD 4 MAB18.4.34 (27-31) gold conjugate

Example 2 Correlation of the Epitope/Antibody Combination MAB 17.3.1(13-16)/PAB (39-50) and MAB 18.4.34 (27-31)/PAB (39-50) in the Elecsys®Format to the Elecsys® NT-proBNP Test Kit (cf., FIGS. 2 and 3)

The correlation of the MAB/PAB combinations MAB 17.3.1 (13-16)/PAB(39-50) and MAB 18.4.34 (27-31)/PAB (39-50) to the Elecsys® test kit(PAB (1-21)/PAB (30-50)) was examined in an electrochemiluminescenceimmunoassay on an Elecsys® 2010 (Roche Diagnostics). For this the PAB(39-50) was used as a biotinylated capture reagent and ruthenylatedF(ab′)₂ fragments of the MABs were used as the detection reagent. 20 μlsample or standard material was in each case incubated with 75 μl of thetwo antibody reagents for 9 minutes at 37° C. Afterwards, 35 μlstreptavidin-coated magnetic polystyrene particles were added and it wasincubated for a further 9 minutes at room temperature. Theelectroluminescence signal of an aliquot of the incubation solution wasmeasured routinely on the Elecsys® 2010 and converted into aconcentration signal by means of a standard curve.

Clinical samples from patients with cardiac failure were now measuredwith the two MAB/PAB test variants and the Elecsys® kit. The results areshown in FIGS. 2 and 3. A very good correlation to the Elecsys® kit(r=0.978 and r=0.957) was obtained with both MAB/PAB variants.

Example 3 Function Curve of an NT-proBNP Test Strip with Two DifferentMAB/PAB Combinations

An NT-proBNP test strip was prepared according to Example 1. Thefollowing impregnation formulation for the reagent fleeces was used:

“biotin fleece”: 100 mM Hepes pH 7.4, 0.1% Tween ®, 20 μg/mlbiotinylated PAB (39-50) “gold fleece”: 100 mM Hepes pH 7.4, OD 4 MAB18.4.34 (27-31) or MAB 17.3.1 (13-16) gold conjugate

Heparinized blood samples from healthy donors were spiked with seracontaining NT-proBNP from heart failure patients and aliquoted. 150 μlof the spiked blood samples was pipetted onto the test strips andmeasured in a CARDIAC Reader® (Roche Diagnostics). The reaction timeafter sample detection was 12 minutes. In order to determine theNT-proBNP concentration of the samples, plasma was centrifuged from onealiquot and measured with an Elecsys® NT-proBNP kit (Roche Diagnostics).Function curves obtained in this manner of the two test strip variantsMAB 17.3.1 (13-16)/PAB (39-50) and MAB 18.4.34 (27-31)/PAB (39-50) areshown in FIG. 4. The variant MAB 18.4.34 (27-31)/PAB (39-50) has aconsiderably steeper standard curve and is thus a more sensitive test.

Example 4 Correlation of an NT-proBNP Test Strip with the ABCombination: Au-MAB 18.4.34 (27-31)/Bi-PAB (39-50) to the Elecsys®NT-proBNP Test Kit

Sera containing NT-proBNP from patients with cardiac failure were addedto heparinized blood samples from healthy donors and aliquoted. 150 μlof these “spiked” blood samples was pipetted onto the test strips andmeasured in a CARDIAC Reader® (Roche Diagnostics) according to thestandard method. Plasma was centrifuged from the same sample andmeasured with the Elecsys® NT-proBNP kit on an Elecsys® 1010 analyticalsystem (Roche Diagnostics). 60 samples were prepared in this manner andmeasured with both systems. FIG. 5 shows the measured values for bothsystems. The correlation is very good at r=0.95.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that may be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation may vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein as preferredor particularly advantageous, it is contemplated that the presentinvention is not necessarily limited to these preferred aspects of theinvention.

What is claimed is:
 1. An immunological test strip device fordetermining human N-terminal pro-brain natriuretic peptide (NT-proBNP)comprising: (a) a sample application zone for receiving a liquid sample;(b) a combination of antibodies, wherein the combination is: i. amonoclonal antibody that specifically binds an epitope of NT-proBNP,wherein the epitope comprises amino acid residues 27-31 of the sequenceof human NT-proBNP; ii. a polyclonal antibody that specifically binds anepitope of NT-proBNP, wherein the epitope is amino acid residues 38-42of the sequence of human NT-proBNP; wherein at least one antibody in thecombination of antibodies is attached to a particulate label selectedfrom the group consisting of a gold label, a colored lattice, a metalsol label, a polymer label, a semiconductor nanocrystal and biotin; and(c) a detection zone in fluid communication with the sample applicationzone for binding a complex comprising the NT-proBNP and the antibodiesof the combination.
 2. The device of claim 1, wherein the sampleapplication zone comprises a matrix material.
 3. The device of claim 2,wherein the matrix material is impregnated with at least one of themonoclonal antibody and the polyclonal antibody.
 4. The device of claim2, wherein the matrix comprises at least two fleece structures.
 5. Thedevice of claim 1, wherein the detection zone comprises at least oneimmobilized immunochemical component.
 6. The device of claim 1, furthercomprising an erythrocyte separation zone in fluid communication withthe sample application zone.
 7. The device of claim 6, wherein theerythrocyte separation zone is in fluid communication with the detectionzone.
 8. The device of claim 1 further comprising a suction zone influid communication with the detection zone.
 9. The device of claim 1wherein the test strip device is an immunochromatographic test device.10. The device of claim 1 wherein the particulate label is a gold label.11. The device of claim 1 wherein the particulate label is biotin.
 12. Amethod for detecting human N-terminal pro-brain natriuretic peptide(NT-proBNP) comprising: (a) providing the immunological test stripdevice of claim 1; (b) adding a biological sample suspected ofcontaining NT-proBNP to the test strip device; and (c) determiningpresence or amount of NT-proBNP in the sample that binds to both theantibodies in the combination of antibodies.
 13. A method fordetermining N-terminal pro-brain natriuretic peptide (NT-proBNP) in ahuman patient sample, the method comprising: (a) contacting a patientsample with an immunological test strip device comprising a combinationof antibodies, wherein the combination is: i. a monoclonal antibody thatspecifically binds an epitope of NT-proBNP, wherein the epitopecomprises amino residues 27-31 of the sequence of human NT-proBNP; ii. apolyclonal antibody that specifically binds an epitope of NT-proBNP,wherein the epitope is amino residues 38-42 of the sequence of humanNT-proBNP; wherein at least one antibody in the combination ofantibodies is attached to a particulate label selected from the groupconsisting of a gold label, a colored lattice, a metal sol label, apolymer label, a semiconductor nanocrystal and biotin; (b) forming acomplex comprising the NT-proBNP from the sample, and the antibodies ofthe combination, wherein one of the antibodies of the Combination isbound to or becomes bound to a detection zone of the test device; and(c) detecting the complex, thereby determining the NT-proBNP in thesample.
 14. The method of claim 13 wherein the sample is whole blood,plasma or serum.
 15. The method of claim 13 wherein the particulatelabel is a gold label.
 16. The method of claim 13 wherein theparticulate label is a biotin label.
 17. An immunological test stripdevice for determining human N-terminal pro-brain natriuretic peptide(NT-proBNP) comprising a test strip comprising: (a) a combination ofantibodies, wherein the combination is: i. a monoclonal antibody thatspecifically binds an epitope of NT-proBNP, wherein the epitopecomprises amino acid residues 27-31 of the sequence of human NT-proBNP;ii. a polyclonal antibody that specifically binds an epitope ofNT-proBNP, wherein the epitope is amino acid residues 38-42 of thesequence of human NT-proBNP; wherein at least one antibody in thecombination of antibodies is attached to a particulate label selectedfrom the group consisting of a gold label, a colored lattice, a metalsol label, a polymer label, a semiconductor nanocrystal and biotin; (b)a sample application zone for receiving a liquid sample, wherein thesample application zone comprises a matrix material impregnated with atleast one of the monoclonal antibody and the polyclonal antibody; and(c) a detection zone in fluid communication with the sample applicationzone for binding a complex comprising the NT-proBNP and the antibodiesof the combination.